The present invention relates to a multi-modality fluorescence reference plate useful for calibrating and testing fluorescence spectroscopic instruments, such as microscopes, imaging devices and plate readers. The invention also relates to a method of manufacturing such plates and to the use of the plates in steady-state fluorescence, time-resolved fluorescence, fluorescence polarisation and fluorescence lifetime.
Many dye molecules, when irradiated with visible or ultraviolet light, emit a portion of the absorbed energy as fluorescent light at longer wavelength. These dye molecules, known as fluorogenic compounds, are widely used in biological assays, where the fluorescent signals they produce can provide information about the system under study. The technique of fluorometry is extremely powerful, since it provides an extremely sensitive measurement on very small quantities of materials under study.
Fluorometers have three principal components: i) a light source for excitation; ii) one or more filters and/or dispersive monochromators for selecting the wavelength of interest; and iii) a detector for converting the fluorescence light into an electrical signal. Traditionally, most detectors have consisted of either a diode or a photomultiplier tube (PMT), both of which measure a single sample at a time. More recently, detectors which comprise a charge-coupled device (CCD) have been used since they enable simultaneous imaging and quantification of many fluorescent samples at one time.
The high sensitivity of fluorometric techniques, and concomitant low sample demands, has made them a favoured screening method for new drug discovery in the pharmaceutical industry where they have found great utility in high throughput screening (HTS). Multi-well or micro-well plates are frequently used in HTS since their compact format (typically 96 or 384 wells in a 126×84 mm footprint) maximises throughput while minimising sample and space requirements. Such plates are well known in the art and are available from a number of commercial suppliers (e.g. Greiner Labortechnik).
The most common form of fluorometers used in HTS are PMT-based scanners, in which one well is measured at a time in a supposedly ‘identical’ manner (e.g. Farcyte™, Amersham Biosciences, Buckinghamshire, UK). Fluorometers of this type are known as plate readers. The process generally involves the plate moving between readings to allow each well to be aligned beneath the detector and the fluorescent signal to be measured from each. Frequent checks are therefore necessary, using standard solutions of ‘known’ fluorescence, to ensure that the system is behaving correctly and that signals from all the wells are being measured in an identical manner.
More recently CCD-based imaging systems, such as Leadseeker™ (Amersham Biosciences) and Viewlux™ (PerkinElmer Life Sciences, Inc., Massachusetts, USA), have been used in HTS applications as they significantly reduce assay time and increase throughput by imaging whole plates simultaneously. The INCell™ 1000 and 3000 Cell Analysers (Amersham Biosciences) are integrated automatic image acquisition and analysis instruments for use in high throughput cell screening assays at the subcellular level. Once again, frequent checks are necessary to calibrate the instrument and ensure that it is performing in a reproducible and accurate manner.
There is therefore a continuing need for calibrating fluorometric instruments by making regular measurements on fluorescent standards. Such standards are based upon properly characterised sources of signals which do not vary significantly from test-to-test or from laboratory-to-laboratory.
Many methods exist in the art for achieving this goal. Thus, for example, Model & Burkhardt (Cytometry, 2001, 44, 309–316) report on a method for normalising fluorescent images to that of an image of a reference standard using stock solutions of fluorescein and microscope slides. This method, however, is not readily applicable to HTS and the use of micro-well plates.
U.S. Pat. No. 6,348,965 discloses a solid state device for the calibration of microplate fluorescence and absorption readers. The invention described consists of a series of optical glass probes coated with a fluorogenic material which are shaped to fit into the wells of a microplate. The user is therefore required to place the appropriate probe, coated with a particular concentration of a fluorogenic compound, manually into specific wells of the microplate. Such an operation can be both time-consuming and prone to errors. Furthermore, the continued handling of the probes can lead to excessive wear of the fluorogenic coating with a resultant reduction in fluorescent signal.
US 2002/0048817 describes standards for calibrating fluorescent instruments which consist of viscosity changing polymers and dyes. The standards can be used to dissolve a wide range of different dyes which are then subsequently dispensed into microwell plates, transformed into gels and used to calibrate the instrument. Once again, this method necessitates manual or mechanical preparation of the solutions and dispensing into micro-well plates.
US 2003/0012702 discloses a fluorescence validation microplate for testing the validity of a fluorometer. Fluorophores of known excitation and emission wavelengths are fitted or placed in the wells of the microplate. The fluorophore can be an organic or inorganic material on the surface of a film, coated or frosted onto a rigid slide, or embedded in a polymer matrix which is inserted into the wells or troughs in the plate. The preparation of such plates can be time-consuming to ensure that the fluorophore is fitted evenly into the well or trough.
Several products are commercially available for calibration of fluorometric devices. In the simplest form, Varian Inc. (Mesa Components, California, USA) provide a fluorescence reference set consisting of fluorescence standard materials in polymer blocks (ref. 66 100 103 00) for use with fluorometric instruments. Starna (Optiglass Ltd., Essex, UK) offer a similar ‘Reference Set’ (ref. 6BF) of stable fluorescent materials in hydrocarbon blocks. Such materials do not lend themselves readily for calibrating PMT fluorimeters and CCD-based imaging systems.
BMG Labtechnologies Ltd. (Buckinghamshire, UK) offer a ‘Calibration Microplate’ (ref. CLS96M) which can be used to measure fluorescence. The microplate relies on LED/solid state technology, in twelve wells, to generate a reproducible light signal in the 500–520 nm range. With only twelve wells capable of generating a signal, the calibration microplate has little utility as a reference standard for CCD-based imaging systems.
The QC Pak™ micro-well plate, supplied by Innovative Instruments Inc. (North Carolina, USA), is suitable for both PMT-based and CCD-based imaging systems. The QC Pak™ product consists of organic fluorophores (e.g. fluorescein, rhodamine, umbeliferon) embedded within a styrene matrix in the wells of an anodised aluminium 96 micro well plate. The plate is ready for use and contains a range of different fluorophores at varying concentrations, thus enabling calibration of both wavelength and intensity. The manufacturing process, however, imposes certain restrictions on the product in terms of cost and the availability of only those dyes that are soluble in organic solvents.
Matech™ (Health Scientific Ltd., Buckinghamshire, UK) provide fluorescence reference standards (‘FRS’) in the form of multi-well plates. Many of the wells of these plates contain a series of inorganic standards, at varying concentrations, which are radioactive in nature and which emit fluorescent signals of specific wavelength and intensity when irradiated by a particular source. Due to the radioactive nature of the inorganic standard, it may be necessary to apply certain safety restrictions when handling or using these plates. Furthermore, the number of reference standards are restricted to those ‘inorganic standards’ commercially available.
There is therefore a need for a cost effective reference plate which can be used in either simple diode/PMT modality or multi-CCD-imaging modality to calibrate fluorescent instruments. The present invention addresses many of the above mentioned limitations of the prior art devices and provides a multi-modality fluorescence plate which can be used to calibrate plate alignment, fluorescence wavelength, intensity and lifetime.